a) Field of the Invention
The present invention relates to screening of nucleic acids. More particularly, the present invention is concerned with the identification of nucleic acids having a desired feature, such as nucleic acids encoding signal peptides, secreted proteins, membrane bound proteins, proteases and drug-resistance proteins.
b) Brief Description of the Prior Art
Large-scale gene sequencing projects are currently generating huge amounts of genetic information. Biomedical research is therefore faced with the challenge of deciphering this genetic information. This is usually done with the help of functional genomic tools. There are currently two main approaches to massively analyze gene function. The first one involves determining the expression profile of genes in normal and diseased tissues. This approach only gives circumstantial evidence to gene function and allows at best the formulation of hypotheses with respect to the function of differentially expressed genes. The other approach is a more biological approach and it uses model organism or cell-based assays to selectively identify genes possessing a chosen biological property or function. Both approaches are complementary and ultimately converge toward the characterization of therapeutically useful genes.
Cell-based screening technology can be viewed as a tool that sends out a positive xe2x80x9csignalxe2x80x9d if, and only if, a particular xe2x80x9ctargetxe2x80x9d gene possessing the activity being screened for has been incorporated into a cell. This technology is based on a reporter system that is kept inactive in the absence of the target gene. Although the nature of the reporter and the conditions upon which it is activated may vary, the reporter systems typically involve the synthesis of either marker proteins (e.g. antibodies), proteins necessary for cell survival under selective conditions (e.g. aminoglycoside3xe2x80x2 phosphotransferase), or fluorescent tags such as green fluorescent protein. In every case, a cell selection procedure is required to identify the gene which has activated the reporter. When screening is conducted with micro-organisms (e.g. Escherchia coli, Saccharomyces cerevisiae), the cell selection step is relatively straightforward. However, mammalian cells are the model of choice to study genes involved in human diseases and, unlike micro-organisms, mammalian cells cannot be easily selected in culture. Methods for selecting mammalian cells either require sophisticated and expensive hardware (e.g. fluorescence-activated cell sorting) or are lengthy and susceptible to high percentages of false-positives due to mutation/adaptation (growth of cells in selective medium). Furthermore, these known methods cannot greatly amplify the exogenous nucleic acid encoding a protein of interest since it gives rise to at most a few cells in which the reporter has been activated.
Viruses have been used for many years to introduce and express exogenous coding sequences in a host cell. Among the viruses used, recombinant Sindbis virus described in U.S. Pat. No 5,217,879 is particularly useful since it can infect cells from various different tissues and express an exogenous coding sequence at relatively high levels. Recently, the scientific community has foreseen the potential of viruses in gene screening methods, particularly as a mean to introduce genes into mammalian cells. International patent application No. PCT/US99/01164 published under No. WO99/36516 describes the use of virus vectors for selecting desired functions of RNAs and proteins. Similarly, International patent application No. PCT/US98/24520 published under No. WO99/25876 describes a method for the detection, characterization and isolation of nucleic acids encoding proteins of a desired property. These methods are based on the expression of high levels of an exogenous protein in a cell and on the marking or observance of the exogenous protein so produced to determine its function. Therefore, the methods described in these two international applications are different from the present invention. Indeed, these international applications do not describe a method wherein viral particles are produced only once an exogenous nucleic acid having a desired property has been inserted into a viral genome. They do not describe either a nucleic acid molecule encoding a dysfunctional viral genome wherein production of a viral particle is dependent on insertion into the dysfunctional viral genome of an exogenous nucleic acid having a desired property such as a nucleic acid encoding a signal peptide, a secreted protein, a membrane protein, a protease or a drug-resistance protein.
In view of the above, it is clear that there is a need for methods and tools which overcome the limits and obviate the problems known in the art. More particularly, there is a need for a gene screening method that bypasses the time-consuming cell selection step and that does not require marking of proteins. The purpose of this invention is to fulfill these needs by taking advantage of the natural property of viruses to incorporate and export genetic material out of a cell. The purpose of this invention is also to fulfil other needs that will be apparent to those skilled in the art upon reading the following specification.
It is therefore an object of the present invention to provide a molecule, a method and a kit for selecting a nucleic acid having a desired feature. More particularly, it is an object of the invention to provide a modified viral genome capable of both expressing exogenous nucleic acids within a host and selecting among these nucleic acids the ones having a desired feature, a predefined characteristic or function. Such nucleic acids include nucleic acids encoding a signal peptide, nucleic acids encoding at least partially for a protein having a signal peptide, nucleic acids encoding proteases, nucleic acids encoding proteins or peptides having a proteolytic activity and nucleic acids encoding drug-resistance proteins or peptides.
In one aspect, the invention is directed to a method for selecting a nucleic acid having a desired feature and the method comprises the steps of:
a) providing a viral genome capable, when present into a suitable host, of expressing an exogenous nucleic acid inserted therein and also capable of packaging itself into a viral particle;
b) providing a suppressive condition wherein the viral genome is capable of packaging itself into a viral particle only once said suppressive condition is being overcome;
c) inserting an exogenous nucleic acid into the viral genome to provide a recombinant viral genome;
d) transfecting the recombinant viral genome into a suitable host; and
e) allowing the recombinant viral genome to express said exogenous nucleic acid and package itself into a recombinant viral particle. The production of at least one recombinant viral particle is then indicative that the suppressive condition has been overcome and that the exogenous nucleic acid inserted in step c) has the desired feature.
Preferably, the suppressive condition is provided by modifying the viral genome in order to inactivate a viral gene product involved in the packaging of the viral particle(s). The viral genome can also be modified to abolish its autoreplicative functions(s) and/or the infectivity of viral particles produced therefrom.
In one embodiment, the viral genome is modified in order to encode a dysfunctional signal peptide and the production of a viral particle is dependent on insertion into said viral genome of an exogenous nucleic acid encoding a functional signal peptide or at least partially a protein having a signal peptide. It is therefore possible to select a nucleic acid encoding a signal peptide or a protein having a signal peptide using the method of the invention. The present invention thus also provides a method for selecting a nucleic acid encoding a signal peptide or a protein having a signal peptide, this method comprising the steps of:
a) providing a viral genome modified in order to encode a dysfunctional signal peptide and wherein production of a viral particle is dependent on the expression of a functional signal peptide;
b) inserting an exogenous nucleic acid into the nucleic acid encoding the dysfunctional signal peptide to provide a recombinant viral genome;
c) transfecting the recombinant viral genome of step b) into a suitable host; and
d) allowing the recombinant viral genome to express the exogenous nucleic acid and package itself into a recombinant viral particle, whereby production of at least one recombinant viral particle is indicative that the exogenous nucleic acid inserted in step b) encodes a signal peptide or a protein having a signal peptide.
In an other embodiment, the viral genome is modified in order to encode a fusion protein comprising a structural viral protein bound to a fetter-protein and the production of a viral particle is dependent on liberation of said structural viral protein from said fetter-protein. It is therefore possible to select a nucleic acid encoding a protease, or encoding a protein or a peptide having a proteolytic activity using the method of the invention. The present invention thus further provides a method for selecting a nucleic acid encoding a signal peptide or a protein having a signal peptide, this method comprising the steps of:
a) providing a viral genome modified in order to encode a fusion protein comprising a structural viral protein bound to a fetter-protein, wherein production of a viral particle is dependent on liberation of the structural viral protein from the fetter-protein;
b) inserting an exogenous nucleic acid into the viral genome of step a) in order to provide a recombinant viral genome;
c) transfecting the recombinant viral genome of step b) into a suitable host; and
d) allowing the recombinant viral genome to express the exogenous nucleic acid and package itself into a recombinant viral particle. The production of at least one recombinant viral particle is then indicative that the exogenous nucleic acid inserted in step b) encodes a peptide or a protein having a proteolytic activity or a protease capable of liberating said structural viral protein from said fetter-protein.
The suppressive condition can also be provided by exposing the transfected host to a substance inhibiting viral packaging function(s) such as cerulenin and okadaic acid. The present invention thus further provides a method for selecting a nucleic acid encoding a drug-resistance protein or peptide, this method comprising the steps of:
a) providing a viral genome encoding a viral particle, said viral genome when present into a suitable host having the abilities of:
i) expressing an exogenous nucleic acid inserted therein; and
ii) packaging said exogenous nucleic acid into a recombinant viral particle;
b) inserting an exogenous nucleic acid into the viral genome to provide a recombinant viral genome;
c) transfecting said recombinant viral genome into a suitable host; and
d) exposing the host transfected in step c) to a substance inhibiting viral packaging function(s),
e) allowing the recombinant viral genome to express the exogenous nucleic acid and package itself into a recombinant viral particle. The production of at least one recombinant viral particle is then indicative that the exogenous nucleic acid inserted in step b) encodes a drug-resistance protein or peptide.
Although, theoretically, any viral genome could be used according to the present invention, viruses of the group of RNA viruses are preferred, and more preferably members of the Alphavirus family such as the Sindbis virus or the Semliki forest virus. Preferably the viral genome used is also capable of autoreplication and insertion therein of an exogenous nucleic acid having a desired feature results in the production of a clonal population of recombinant viral particles. It is also preferable to incorporate the viral genome into a vector such as a bacterial plasmid in order to facilitate the insertion of exogenous nucleic acids and the modification of the viral genome.
In another aspect, the invention is directed to an isolated nucleic acid molecule encoding a dysfunctional viral genome. Using the isolated nucleic acid molecule of the invention, the production of a viral particle is dependent on: 1) insertion of an exogenous nucleic acid having a desired feature into the nucleic acid molecule of the invention; and 2) introduction of this nucleic acid molecule incorporating the exogenous nucleic acid into a suitable host.
In a further aspect, the invention is directed to a kit for selecting a nucleic acid having a desired feature, this kit comprising:
a nucleic acid molecule encoding a dysfunctional viral genome according to the present invention; and
at least one further element selected from the group consisting of instructions for using said kit, reaction buffer(s), enzyme(s), probe(s) and pool(s) of exogenous nucleotide sequences.
Also encompassed within the invention is a N-terminal amino acid sequence encoding a dysfunctional signal peptide of a viral envelope protein. This dysfunctional signal peptide has the characteristics of allowing viral envelope proteins association without directing said viral envelope protein into the cellular secretory pathway and across the lipid bilayer of a host cell.
An advantage of the present invention is that it takes advantage of the natural property of viruses to export genetic material out of a cell. Accordingly, it obviates the expensive and time-consuming task of selecting cells that express a gene of interest. The invention is also much more rapid, efficient and accurate for selecting a particular nucleic acid having a desired feature, characteristic or function. The invention can also selectively retrieve from a library of nucleic acids a nucleic acid having a desired feature among a pool of nucleic acids.
Other objects and advantages of the present invention will be apparent upon reading the following non-restrictive description of several preferred embodiments, made with reference to the accompanying drawings.